CA2067317A1 - Caffeoyl-coa 3-o-methyltransferase genes - Google Patents

Abstract

Caffeoyl-CoA 3-0-methyltransferase genes A b s t r a c t The present invention relates to new caffeoyl-CoA 3-0-methyltransferase genes isolated from plants and their use for the transformation of vectors, host organisms and plants and for the generation of plants which have an increased resistance to pests.

Le A 28 417

Description

2~673~7 The present invention rela~es to new caffeoyl-CoA 3-o-methyltransferase genes (called CCoAMT genes below) isolated from plants and to their use for the transforma-tion of vectors, host organisms and plants and for the S generation of plants which have an increased resistance to pests.

The enzyme caffeoy~-CoA 3-O-methyltransferase, called CCoAMT below, catalyses the methylation of caffeoyl-CoA
in a biosynthesis route, which has only recently been described, which leads from trans-4-coumaroyl-CoA to trans-feruloyl-CoA (Matern, U., and Kneusel, R.E. 1~88, Phytoparasitica 16:153-170; Kneusel, R.E., Matern, U., and Nicolay, K. 1989, Arch. Biochem. Biophys. 269:455 to 462; and Pakusch, A.-E., Rneusel, R.E., and Matern, U., lS 1989, Arch. Biochem. Biophys. 271:488 to 494).

Under fungal attack, plants reinforce their cell wall very rapidly by incorporation of cinnamic acids, followed by cross-linking thereof to give polymeric structures or build-up of lignin. Under these conditions, feruloyl-CoA
is the preferred acyl donor both for the esterification of cell wall polysaccharide~ and for lignification (reduction to coniferyl alcohol). The speed and extent of the change in the cell wall essentially determine the course of the infection and the fate of the plants, 2S "hypersensitive reaction" characterising complete resis-tance of the plants, associated with a particularly severe and rapid change in the cell wall and the death of the cells directly affected. This hypersensitive reaction is also observed in the resistance reaction of plants to Le A 28 417 - 1 -virus infections. It has only recently been discover~d~ 7 317 that feruloyl-CoA is not formed in vivo in all cases by activation of ferulic acid, but is also formed by reac-tion of coumaroyl-CoA. The caffeoyl-CoA-specific methyl-transferase which participates in this reaction hasscarcely any homology with previously known enzymes (Pakusch, A.-E., Matern, U., and Schiltz, E., 1991, Plant Physiol. 95:137 to 143), is taxonomically widespread in plants and can be induced therein by, for example, fungal 1~ attack.

A large proportion of the world harvest of crop plants is constantly destroyed by pests (in 1967 the loss of potential harvest was 35%; compare Chemistry of Pesti-cides, published by ~.H. Buchel, John Wiley & Sons, New York, 1983, page 6). There is therefore an urgent need to research and utilise all possibilities which are capable of reducing or preventing attack of crop plants by pests.

The new caffeoyl-CoA 3-0-methyltransferase genes, called CCoAMT genes below, have now been found, which can be incorporated into the hereditary factors ~the genome) of plants which generate no CCoAMT or only inadequate CCoAMT, whereby an increased resistance of these plants topests can be brought about.

It is surprising that it has bee!n possible to find a new type of resistance genes which can be incorporated as foreign or additional DNA into the genome of plants, whereby an increased resistance of the resulting trans-genic plants to pests .is achieved. A particular advantage of the present invention is that - in contrast to, for example, the case of increased accumul2tion of Le A 28 417 - 2 -phytoalexins - it is not aimed at the generation ~ ~ 7 3 ~ 7 potentially toxic metabolites. There are th~refore also no toxicological reservations, because the aim is the rapid s~lthesis in the transformed plants of predominantly insoluble, antibiotically inactive compounds which should function as physical barriers or prevent possible pathogen-induced, enzymatic lysis of cell wall polysaccharides by acylation of the "substrate~. In contrast to the transformation of plants with genes of lytic enzymes, such as, for example, lysozyme or also chitinase, which at best can become selectively active, the increased readiness of plants to reinforce the cell wall offers protection against every form of pathogens, including viruses. The present invention here therefore follows a novel principle of plant protection with wide application.

By CCoAMT genes, there are to be understood any nucleic acid (DNA) which, after its transcription into RNA and translation into protein, causes the formation of an enzyme which has the properties of a CCoAMT, this nucleic acid being isolated from its natural environment or integrated into a vector or contained as "foreign" DNA or as "additionalll DNA in a prokar.yotic or eukaryotic DNA.
By CCo~MT genes there are also to be understood those ~5 CCoAMT genes which contain, at their start and/or end, ad-ditional DNA sequences which do not or do not substantial-ly impede the function of the genes. These DNA sequences, which are also called "gene units", are formed, for example, by excision with restriction enzymes, since no cleavage sites are available for customary restriction enzymes exactly at the start and at the and of the gene.
The CCoAMT genes or the gene units can also carry at Le A 28 417 - 3 -2~3~ 7 their ends ~NA sequences which are appropriate for their handling (for example "linkers").

The CCoAMT genes tox the gene units) can exist in the form in which they are contained in the genome of plants S ("genomic" form, including sequences which do not encode CCoAMT and/or do not have a regulatory action (such as introns~), or in a form which corresponds to the CDNA
(~'copy~' DNA~ which is obtainable via mRNA with the aid of reverse transcriptase/polymerase (and no longer contains introns). r~ e ~oAMT genes can also be present in par-tially or completely synthetic form. By synthetic genes there are also understood those which are formed by newlyjoining of parts of natural genes.

DNA segments or DNAs in the CCoAMT genes (or the geneunits) according to the invention can be replaced by other DNA
segments or DNAS which have essentially the same action.

In the present connection, by "foreign" DNA there is to be understood DNA tin particular genes or gene units or componenents thereof) which does not occur naturally in a certain prokaryotic or ~eukaryotic genome, but is taken up in this genome only as a result of intervention by man. ~'Additional" DNA (in particular genes or gene units or components t~ereof) is intended to mean DNA
whicht although it occurs naturally in the particular prokaryotic or euka~yotic genome, has been taken up in this genome in an additional amount as a result of in~ervention by man. One or more copies of the "foreign"
DNA or "additional" DNA can be incorporated, depending on requirements and on the nature of the case in question.

Le ~ 28 417 - 4 -2~3~
CCoAMT which is formed in plants or plant cells with the assistance of the CCoAMT genes (or the gene units) according to the invention means any enzyme which acts like CCo~MT and, in plants, increases their resistance to pests.

The preferred CCoAMT genes according to the invention are characterised in that they hybridise with the CCoAMT-cDNA
sequence contained in the plasmid pL2-4 or its components or with the cDNA sequence according to SEQ ID No: 1 or its components and encode CCoAMT.

CCoAMT genes which are preferred according to the inven-tion are the CCoAMT genes which occur in parsley (Petro-selinum crispum), carrots (Daucus carota), carnation (Dianthus caryophyllus) and safflower (Carthamus tincto-rius), particularly preferably in parsley, and can be isolated from these.
The CCo~MT gene which is present (as a gene uni~) in the form of the cDNA on the plasmid pL2-4 (which is described below in more detail) and the DNA sequences which have essentially the same action are especially preferred as the CCoAMT gene according to the invention.

The cDNA contained on the p].asmid was isolated from parsley. It consists of a 5' unt:ranslated leader sequence 370 nucleotides long and the complete protein-encoding region from position 371 to position 1093, followed by 67 nucleotides of a 3' untranslated sequence. The entire frasment was provided with EcoRI linkers on both sides and cloned into the vector pGEM 7 (Promega Corp. Madison, Wi., USA). The residual sequence of the 3' untranslated region from position 1160 to 1258 is not present on the Le A 28 417 - 5 _ . .. _ _ .. _ _ _ . 1.~ . . .. _ ~ _ , . '' A .: '.~

~7317 plasmid pL2-4. This poly-adenylation sequence can be prepared synthetically or replaced by another poly-~sequence. The complete cDNA sequence can be seen from sequence protocol SEQ ID No:1.

The 5' untranslated region, the complete encoding region and 67 nucleotides of the 3~ untranslated region can be isolated in the customary manner with ~coRI on a fragment about 1170 long.

The chimaeric gene fusions of the TR promoter or the 35S
promoter with the protein-encoding region of the CCoAMT
genes, preferably of the gene from parsley, inparticular of the gene which corresponds to the cDNA on the plasmid pL2-4, may be mentioned as particularly preferred.
It has been found that the CCoAMT genes which occur in plants have wide regions of DNA sequence homology. On the basis of the sequence homoloqy, the CCoAMT genes accord-ing to the invention can therefore be isolated from plants in a simple manner with the aid of the cDNA
contained on the plasmid pL2-4 or its components or the sequence information according to SEQ ID No: 1 in the customary manner using the known methods of molecular biology.

Possible plants from whi~h CCohMT genes according to the invention can be isolated are practically all the mono-2S cotyledonous or dicotyledonous plants, preferably dicoty-ledonous plants, parsley, carrot, safflower and carnation being mentioned by way of example and as preferred.

As already mentioned, the CCoAMT gene, or the encoding region thereof, which corresponds to the cDNA which lies Le A 2~ 417 - 6 -~0~73~7 on the plasmid pL2-4 is preferred according to the inven-tion. The gene or the coding region of the gene can be obtained in the customary manner with the aid of the cDNA.

The Escherichia coli s~rain DS pL2-4 contains the plasmid pL2-4. This strain has been deposited at the Deutsche Sammlung von Mikroorganismen (DSM3 rGerman Collection of Microorganisms], Mascheroder Weg lb, D-3300 Braunschweig, Federal Republic of Germany, in accordance with the conditions of the Budapest Treaty on the International Recognition of Deposition of Microorganisms for the Purposes of Patent Proceedings (deposition date- 28th May 1991). It has been given deposition number DSM 6~36.

The present invention also relat~s to this strain and its mutants. The plasmid pL2-4 deposited in this host can easily be obtained in the required amoun~s in the customary manner by multiplication of the strain and subsequent isolation of the plasmid.

Functionally complete genes, ~iuch as the CCoAMT genes according to the invention, consist of a component which has a regulatory action (in particular a promoter) and the structural gene which codes for the protein CCoAMT.

Both parts of the gene can be used independently of one another. It is thus possible to fuse the component having the regulatory action wi~h another DNA sequence (deviating from the CCoA~T gene) which i5 to be expressed after incorporation into the plant genome. Since only a few isolated promoters which can display their action in plants or plant cells are known, the promoters of the Le A 28 417 - 7 -. ~

2~67~ 1 7 CCoAMT genes, to which the present invention likewise relates, are useful aids in the generation of transformed plants or plant cells.

It is also possible to have the CCoAMT structural genes preceded by a "foreign" compon~nt having a regulatory action. This could be advantageous if only specific regu-latory active gene components (for example those endogenous to the plant) can have a sufficient action in certain plants. The CCoAMT structural genes are therefore valuable units which can be used independently and, as already mentioned, the present invention also relates to them.
The cco~r genes according to the invention can be separated into the components having a regulatory action and the structural genes by the customary methods. It is also possible to combine components of different natur-ally occurring CCoAMT genes to give new functional "synthecic" genes. The complete naturally occurring CCoAMT genes (or the gene units) according to the invention are preferably used. The CCoAMT structural gene which corresponds to the cDNA contained in the plasmidpL2-4 is preferred according to the invention.

It is possible, with the aid of customary methods, to incorporate the CCoAMT genes (or the gene units) or their components in one or severa:L copies (for example in tandem arrangement), preferably once, into any desired prokaryotic (preferably ~acterial) or euka~yotic (prefer-ably plant) DNA as "foreignl' or "additional" DNA. Thus, for example, the protein-encoding DNA corresponding to the cDNA can be provided wi~h regulatory sequences and incorporated into plants. The present invention relates to the recombinant DNA "modified~ in this way, which can ., ~ Le A 28 41? - 8 -:;

2~673~7 be used, for example, for the transformation of plants or plant cells and is contained in the plants or plant cells after the transformation.

The CCoAMT genes (or the ~ene units) and/or their compon-ents and the recombinant DN~ can be contained as ~foreign~ or ~additional~ DNA in vectors tin particular plasmids, cosmids or phages), in transformed micro-organisms (preferably bacteria, in particular Gram-negative bacteria, such as E. coli) and in transformed plant cells and plants or in the DNA thereof. The presant invention relates to such vectors, transformed micro-organisms (which can also contain these vectors) and the transformed plant cells and plants and DNA thereof.

AS already indicated, according to the invention the CCoAMT genes (or the gene units) are incorporated in on~
or several copies (at the same or different points ofthe genome) into the natural plant genome, it also being possible for different CCoAMT genes to be combined with one another. In the case of plants which already havethe capacity for CCoAMT synthesis, the incorporation of one or more CCoAMT genes according to the invention can lead to considerably improved resi~tance properties. In the case of plants which contain no CCoAMT genes, an increased resistance to;pests is likewise achieved by incorporation of such genes. If appropriate, only the structural genes according to the inven~ion are used, the~e being preceded by a regulatory DNA element which may have been isolated from the particular plantO

- The increased resistance of the transformed plant cells and plants according to the invention is of Lmportance Le A 28 417 - 9 -2~673~7 for agriculture and forestry and for cultivation of ornamental plants, cultivation of medicinal plants and plant breeding. It is also advantageous in the culture of plant cells, for example for the production of pharma-ceutically usable substances, to have available plant cells which have increased resistances to attack by microbial pests, in particular fungi.

The present invention thus also relates to a process for the preparation of transformed plant cells (including protoplasts) and plants (including plant parts and seeds) having an increased resistance to pests, which is charac-terised in that (a) one or more CCoAMT genes (or gene units) and/or components of the CCoAMT genes (or o the gene units) and/or recombinant DNA according to the invention are inserted into the genome of plant cells (including protoplasts), and if appropriate ~b) complete transformed plants are regenerated from the transformed plant cells (including protoplasts) and if appropriate propagated, and if appropriate (c) the desired plant part,s (including ~eeds) are obtained from the resulting transformed plants of the parent generation or further generations obtained therefrom.

Process steps (a~, (b) and (c) can be carried out in the customary manner by known processes and methods.

The present invention also relates to transformed plant Le A 28 41? - lO -.~, _ . . .

~7~17 cells (including protoplasts) and plants (including plant parts and seeds) which contain one or more CCOAMT genes (or gene units) and/or components of the CCoAMT genes (or of the gene units) as "foreign" or "additional" DNA, and to those transformed plant cells and plants which are obtainable by the above processes.

The present invention also relates to the:

(a) use of the CCoAMT genes (or of the gene units) and/or their components and/or the recombinant DNA
according to the invention and/or the recombinant vectors according to the invention and/or the trans-formed microorganisms according to the invention for the transformation of plant cells (including proto-plasts) and plants (including plant parts and lS seeds), the (b) use of the transformed plant cells (including proto-plasts) and plants (including plant parts and seeds) according to the invention for the generation of propagation material and for the generation of new plants and propagation mat:erial thereof, the (c) use of the CCoAMT genes according to the invention (or of the gene units) andJor their components and/or the recombinant DNA according to the inven-tion for combating pests and the 2~ d) use of the cDNA contained on the plasmid pL2-4 or its components and of the DNA sequences correspond-ing to the sequence information according to sequence protocol SEQ ID NO:1 for isolation of Le A 28 417 ~731~
CCoAMrr genes or components thereof from plants and for the determination of CCoAMT genes in plants.

Ther~ are a number of different methods available for inserting the CCoAMT genes or the gene units or their components into the genetic material of plants or plant cells as "foreign" or ~additional~ DNA. The gene transfer can be carried out by the generally customary known methods, the expert being able to determine without difficulty the particular method suitable.

The Ti plasmid from Agrobacterium tumefaciens is avail-able as a particularly fa~ourable and widely applicable vector for ~he transfer of foreign DNA into genomes of dicotyledonous and monocotyledonous plants. The genetic material which encodes CCoAMT is inserted into the T-DNA
of suitable Ti plasmids together with regulatory ~NA
sequences (for example Zambryski et al. 1983) and trans-ferred by infection of the plants, infection of plant parts or plant tissues, such as, for example, of leaf discs, stems, hypocotyls, cotyledons, meristems and tissues issuing therefrom, SUC}l as, for example, second-ary embryos and calli, or by coculture of protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA which contains the desired gene in plant protoplasts (for example Hain et al., 1985; Rrens et al., 1982; Paszkowski et al., 1984) in the presence of polycations or calcium salts and polyethylene glycol.

The DNA uptake can also additionally be promoted by an electric field (electroporation) (for example Fromm et ., .
Le A 28 417 - 12 -,~

, 3 ~ 7 al., 1986).

The DNA can also be introduced in a known manner via plant pollen, by 'Ishooting" the pollen with physically accelerated particles which carry the DNA (compare EP A
0,270,356).

The plants are regenerated in a known manner with the aid of suitable nutrient media (for example Nagy and Maliga 1976).

In a preferred embodiment of the process according to the in~en~ion, the cDN~ from the plasmid pL2-4 is cloned into an expression vector (for example pRT101, Topfer et.al.
1988). The chimaeric constructed gene is then isolated with the restriction enzyme Hind III and transferred in an intermediate vector (for example pCV001, Roncz and Schell 1986) to Agrobakterium tumefaciens (Koncz and Schell 1986).

Alternatively, the chimaeric constructed gene is cloned into the Hind III position of the plasmid PlGVneo 1103 (Hain et. al. 1985), and in a particularly preferred embodiment the chimaeric const:ructed gene in the plasmid pLGVneo 1103 is transferred in the customary manner to plant protoplasts by direct ~ene transfer (for example Hain et. al. 1985). The plasmid can be in circular form, but is preferably in linear form here.

If this plasmid is used with a reporter gene, the kana-mycin-resistant protoplasts are then checkQd for expres-- ~ion of CCoAMT.
,:
.

~ . Le A 28 417 - 13 -2~67~ 7 Transfoxmed (transgenic) plants or plant cells are generated by the known methods, for example by leaf disc transformation (for example Horsch et al. 1985) by coculture of regenerating plant protoplasts or cell cultures with Agrobacterium tumefaciens (for example Marton ~t al. 1979, Hain et al. 1985) or by direct DNA
transfection. Resulting transformed plants are detected either by selection for expression of the reporter gene, for example by phosphorylation of kanamycin sulphate in vitro (Reiss et al. 1984; Schreier et al. 1985) or by the expression of nopaline synthase (according to Aerts et al. 1983) or CCoAMT by Northern blot analysis and Western blot analysis. The CCoAMT can also be detected in a known manner with the aid of specific antibodies in transformed plants.

Culture of the ~ransformed plant cells and regeneration to give complete plants are carried out by the generally customary methods with the aid of the particular suitable nutrient media.
Both ~he transformed plant cells and the transformed plants which contain the CCoA~ genes according to the invention (or the gene units) and to which the present invention relates exhibit a considera~ly higher resis-tance to pests, in particular phytopathogenic fungi.

In connection with the present invention, the term "plants" denotes both complete plants and also parts of plants, such as leaves, seeds, tubers, cuttings and the like. ~Plant cellsl~ include protoplasts, cell lines, plant calli and the like. ~Propagation material~ denotes plants and plant cells which can be used for propagation of the transformed plants and plant cells, and the Le A 28 417 - 14 -2~73i7 present invention thus also relates to this material.

In the present connection, ~he term DNA sequences having essentially the same action" means that the invention also relates to those modifications in which the function of the CCoAMT genes and their components is not impaired such that CCoAMT is no longer formed or the regulatory gene component is no longer ac~ive. Corresponding modifi-cations can be made by replacement, addition and/or removal of DNA sections, individual codons and/or indi-vidual nucleotides.

In the case of microorganisms which can be used accordingto the invention, "mutants" denotes those modified micro-organisms which still have the features ess~ntial for Lmplementation of the invention, and in particular contain the particular plasmids.
The plants which can be given resistance or an increased resistance to pests by incorporation (transformation) of the CCoAMT genes according to the invention (or the gene units) include practically all plants. $here is of course a particular need for generating resistance in crop plants, such as forest plants, for example spruce, fir, Douglas fir, pine, larch, bee!ch and oak, as well as plants which supply foodstuffct and raw materials, for example cereals (in partIcular wheat, rye, barley, oats, millet, rice and maize), potatoes, leguminous plants (such as pulses and in particlllar alfalfa and soy~eans), vegetables (in particular cabbage varieties and tomatoes), fruit (in particular apples, pears, cherries, grapes, citrus fruits, pineapples and bananas), oil palms, tea, cacao and coffee shrubs, tobacco, sisal and cotton, and in medicinal plants, such as Rauwolfia and Le A 28 417 - 15 -~7317 Digitalis. Potatoes, tomatoes and leguminous plants may ~e mentioned particularly preferably. The CCoAMT genes according to the invention are preferably incorporated into the genome of plants as "foreign-- DNA.

S As pests against which resistances or increased resist-ances can be achieved with the aid of the CCoAMT genes according to the invention there may be mentioned animal pests, such as insacts t mites and nematodes, as well as microbial pests, such as phytopathogenic fungi, bacteria and viruses. Microbial pests, in particular phytopatho-genic fungi, are par~icularly singled out.

The harmful insects include, in particular, insects of the orders:

Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heterop-tera, ~omoptera, Lepidoptera, Coleoptera, Hymenoptera and Diptera.

The harmful mites include, in particular:
Tarsonemus spp., Panonychus spp. and Tetranychus spp.

The harmful nematodes include, in particular:
Pratylenchus spp., Heterodera spp. and Meloidogyne spp.

The microbial pests include, in particular, the phyto-pathogenic fungi:

Plasmodiophoromycetes, Oomyce~es, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deutero-mycetes.

Le A 28 417 - 16 -. . ., . ,. .. ., . ~ ., ~ .

2 ~ 7 The phytopathogenic bacteria include, in particular, the Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae and Streptomycetaceae.

The virus diseases include, in particular, mosaic, S dwarfing and yellowing viroses.

Some causative organisms of viral, fungal and bacterial diseases which come under the generic names listed above may be mentioned as examples, but not by way of limita-tion:
barley yellow dwarf virus (BYDV), potato virus Y ~PVY), cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), q'risteza virus~ tobacco mosaic virus ~TMV), tobacco necrosis virus (TNV~, ~eet necrotic yellow vein virus (BNYVV), rhizomania virus.

Xanthomonas species, such as, for example, Xanthomonas campestris pv. oryzae;
Pseudomonas species, such as, for example, Pseudomonas syringae pv lachrymans;
Erwinia species, such as, for exampler Erwinia amylovora;
Pythium species, such as, for example, Pythium ultimum;
Phytophthora specie~, such as, :for example, Phytophthora infestans;

Pseudoperonospora species, such as, for example, Pseudo-peronospora humuli or Pseudoperonospora cubense;
Plasmopara species, such as, for example, Plasmopara viticola;
Peronospora species, such aq, for example, Peronospora pisi or P. brassicae;

Le A 28 417 - 17 -2~67~17 Erysiphe species, such as, for example, Erysiphe graminis;
Sphaerotheca species, such as, for example, Sphaerotheca fuliginea;
Podosphaera species, such as, for example, Podosphaera leucotricha;
Venturia species, such as, for example, Venturia inaequalis;
Pyrenophora species, such as, for example, Pyrenophora teres or P. graminea (conidia form: Drechslera, syn: Helminthosporium~;
Cochliobolus species, such as, for example, Cochliobolus sativus (conidia form: Drechsler~, syn: Helminthosporium);
Uromyces species, such as, for example, Uromyces appen-diculatus;
Puccinia species, such as, for example, Puccinia recon-dita;
Tilletia species, such as~ for example, Tilletia caries;
Ustilago species, such as, for example, Ustilago nuda or Ustilago avenae;
Pellicularia species, such as, for example, Pellicularia sasakii;
Pyricularia species, such as, fox example, Pyricularia oryzae;
Fusarium species, such as, for example, Fusarium culmorum;

Botrytis species, such as, for example, Botrytis cinerea;
Septoria species, such as, for example, Septoria nodorum;
Leptosphaeria species, such as, for example, Leptosphaeria nodorum;
- Cercospora species, such as, for example, Cercospora Le A 28 417 - 18 -2~73~7 canescens;
Alternaria species, such as, for example, Alternaria brassicae; and Pseudocercosporella species, such as, for example, S Pseudocerco sporella herpotrichoides. ~elminthosporium carbonum may furthermore be mentioned.

The present in~ention shall be illustrated in more detail with the aid of the following embodiment examples:

1. Isolation of the qene for CCoAMT from ~arsleY

Plants and cell cultures from parsley (Petroselinum crispum) contain the genes for CCo.~MT which cause the formation of CCoAMT (size of the pxotein 27,000 D; reac-tion with specific antiserum).

The ~nown processes and methods of molecular biology such as are described in detail, for example, in the following handbook were used in the isolation of the CCoAMT genes:
Maniatis, T., Fritsch, E.F., Sambrook, J.: Molecular Cloning: A Laboratory Manual; C:old Spring Harbor Labora-tory, Second Edition 1989.

A "gene library" for parsley is first established:
genomic DNA from enriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter 2, 24, 1981) is cut with the restriction enzyme NdeII such that DNA fragmen~s having an average length of about 12,000 nucleotide pairs axe formed. These fragments are cloned into the BamHI
site of the lambda phage EMBL4 (Frischauf et al., J. Mol.
Biol. 170, ~27-842, 1983), and the phages are multiplied in E. coli. The phage population in its entirety Le A 28 41? - 19 -2~673~7 contains, cloned in sub fragments, the total genomic DNA
of parsley, and therefore also the genes for CCoMAT.

The genes for CCoMAT, their mRNA and the CCoMAT synthase cDNA each contain the same nucleic acid sequences, since they can be derived from one another (gene ~ mRNA , cDNA). This means that the genes for CCoMAT can be identified by specific hybridisation with CCoMAT-cDNA
(compare SEQ ID NO:l) or with specific oligonucleotides which can be derived from this sequence. The phages with the genes are identified by hybridisation, and then isolated and multiplied. The genomic DNA from parsley cloned in this phage is mapped further by analysis with various restriction enzymes, and the position of the CCoMA~ genes is determined by further hybridisation experiments with cDNA sequences or synthetic oligonucleotides. Finally, the gene units are cut out of the phage by digestion with restriction enzymes, cloned in the correspondingly cut plasmid vector and multiplied as recombinant plasmids.

Because of the sequence homologies, DNA sequences which correspond to the sequences contained in the cDN~ on the plasmid pL2-4 can be used as probes for isolation of other CCoAMT genes according to the inven~ion.

2. Transformation of tobacco a) Culture of tobacco shoots and isolation of tobacco protoplasts:

Nicotiana tabacum (Petit Havanna SRl) is propagated as a sterile shoot culture on hormone-free LS medium Le A 28 417 - 20 -2~7~ ~ 7 (Linsmaier and Skoog 1965). Shoot sections are transferred to fresh LS medium at intervals of about 6-8 weeks. ~he shoot cultures are kept in 12 hours of light (1000-3000 lux) in a culture room at 24-26C.

For the isolation of leaf protoplasts, about 2 g of leaves (about 3-5 cm long) are cut into small pieces (o.s cm x 1 cm) with a fresh razor blade. The leaf material is incubated in 20 ml of enzyme solution consisting of R3 medium (Nagy and Maliga 1976), 0.4 M sucrose, pH 5.6, 2% of Zellulase RlO (Serva) and 0.5% of Nacerozym R10 (Serva) at room temperature for 14-16 hours. The protoplasts are then separated from cell residues by filtration over a 0.30 mm and 0.1 ~m steel sie~e. The filtrate is centrifuged at lO0 x g for lO minutes. During this centrifugation, intact protoplasts float and collect in a band at the top margin of the enzyme solution.
~he pellet of cell residues and the enzyme solution are sucked off with a glass capillary. The prepuri-fied protoplasts are made up to lO ml with fresh R3 medium (0.4 M sucrose as an osmotic agent) and floated again. The washing medium is sucked off and the protoplasts are diluted to 1-2 x 105/ml for culture or subseque~t ini.ection with Agrobacteria (coc~lture)~ The pro~oplast concen~ration i5 deter-mined in a counting chamber.
b) Construction of a chLmaeric CCoAMT gene and transfer into Agrobacterium tumefaciens 30- The EcoRI fragment from pL2-4 (about 1.2 kb) is cloned into the EcoRI position of the vec~or pRT 101 Le A 28 417 - 21 ~

3 ~ 7 (Topfer et.al. 1988). This gives the cDNA the 35 S
promoter of CaMV on its 5' end and a polyadenylation sequence from CaMV on its 3~ end. This chimaeric constructed gene can then be isolated functionally as a fragment of about 1.9 kb ky cleavage with HindIII. This HindIII fragment can then be transferred into an intermediate vector, for example pCV001 ~Koncz and Schell, 1986) by the customary methods. Instead of the vectors mentioned, any other desired expression vectors and intermediate vectors which have corresponding cleavage sites can be employed, the expert easily being able to make a suitable choice on the basis of the above information. The resulting intermediate vector, which contains the CCoAMT gene, is transferred to Agrobacterium tumefaciens which contains a functional vir region (Koncz and Schell 1986, van Haute et.al. 1983).

c) Transformation of regenerating tobacco protoplasts by coculture with Aqrobacterium tumefaciens-The method of Marton et al. 1979 is used below, with minor modifications. The protoplasts are isolated as described and incubated in aldensity of 1-2 x 105/ml in K3 medium (0.4 M ~ucrose, 0.1 mg/l of NAA, 0.2 ml in K3 medium (O.4 M sucrose, 0.1 mg/l of NAA, 0.2 mg of kinetin) for 2 days in the dark and one to two days under weak light (500 lux) at 26C. As soon as the first divisions of the protoplasts occur, 30 ~1 of an Agrobacterium suspension according to b) in minimal A (Am) medium (density about 109 Agro-bacteria/ml) are added to 3 ml of regenerating Le A_28_417 - 22 -2~7~ 7 protoplasts. The duration of the cocul~ure is 3-4 days at 20C in the dark. The tobacco cells are then introduced into 12 ml centrifuge tubes, diluted to 10 ml with seawater t600 mOsm/kg) and pelleted at 60 x g for 10 minutes. This washing operation is repeated a further 1-2 times in order to remove the majority of the Agrobacteria. The cell suspension is cultured in a density of 5 x 104/ml in K3 medium (0.3 M sucrose) with 1 mg/l of NAA (naphthyl-l-acetic acid), 0.2 mg/l of kinetin and sno mg/l of the cephalosporin antibiotic cefotaxLme. The cell suspension is diluted with fresh K3 medium every week and the osmo~ic value of the medium is reduced gradually by 0.05 M sucrose (about 60 mOsm/kg) per lS week. Selection with kanamycin (100 mg/l of kana-mycin sulphate (Sigma), 660 mg/g of active km) is starte~ 2-3 we~ks after the coculture in an agarose ~bead type culture~ (Shillito et al. 1983). Kana-mycin-resistant colonies can be distinguished from ~0 the background of retarded colonies 3-4 weeks after the start of the selection.

d) Direct transformation of tobacco protoplasts with DNA. Calcium nitrate-PEG tra~!sformation.

About 106 protoplas~s in 180 ~1 of K3 medium are carefully mixed in a Petri dish with 20 ~1 of aqueous DNA solution which contains 20 ~g of plasmid pCV001::CCoAMT tcompare Figure 3). The plasmid pCV001::CCoAMT is obtainable by kno~n methods from the plasmid pCV001, pRT101 and pL2-4 (compare Figures 1-3). 200 ~1 of fusion solution l0.1 M
calcium ~litrate, 0.45 M mannitol, 25~ of L~ A 28 417 - ~3 -2a~73~
polyethylene glycol (PEG 6000), pH 9) are then carefully added. After 15 minutes, 5 ml of washing solution (O.275 M calcium nitrate pH 6) are added, and after a further 5 minutes the protoplasts are transferred into a centrifuge tube and pelleted at 60 x g. The pellet is taken up in a small amount of K3 medium and cultured as described in the next section. Alternatively, the protoplasts can be transformed as described by Hain et al. 1985.

e) Culture of the protoplasts incubated with DNA and selection of kanamycin-resistant calli:

A modified "bead type culturel~ technique (Shillito et al. 1983) is used for the culture and selection of kanamycin-resistant colonies described below. One week after treatment of ~he protoplasts with DNA
(compare d~, 3 ml of the cell suspension are mixed with 3 ml of K3 medium (0.3 M sucrose + hormones;
1.2~ (Seaplaque) of LMT agarose (low melting agar-ose, Marine Colloids) in 5 cm Petri dishes. For this purpose, the agarose is autoclaved in ~he dry state and, after addition of K3 medium, is boiled up briefly in a microwave oven. After the agarose has solidified, the agarose discs ("beads") are trans-ferred into 10 cm Petri dishes with the embedded tobacco microcalli for further culture and selec-tion, a~d in each case 10 ml of g3 medium (O.3 M
sucrose, 1 mg/l of NAAI 0.2 mg/l of kinetin) and 100 mg/l of kanamycin sulphate (Sigma) are added.
The liquid medium is changed every week. During this procedure, the osmotic value of the medi~m is reduced in stages.

Le A 28 41? - 24 -~731 7 The replacement medium (K3 + hm) is reduced by 0.05 M of sucrose (about 60 mOsm) per week.

Timeta~le of the selection of kanamycin-resistant tobacco colonies after DNA transformation:

Le A 28 417 - 25 -2~73~ 7 0.4 M 0.3 M 0.25 M 0.20 M 0.1'; M 0.10 M sucrose in the liquid medium A E S K
-1 2 3 4 5 6 weeks after DNA
Uptake (K3 medium 1 mg of NAA, 0.2 mg of kinetin) A = DNA uptake E = embedding in agarose S = selection with kanamycin (100 mg/l of kanamycin sulphate) K = kanamycin-resistant colonies can be clearly distin-guished from the background e~ Reqeneration of kanamycin-resistant plants:

As soon as the kanamycin-resistant colonies have ~0 reached a diameter of about 0.5 cm, half of them are placed on regeneration medi~ (LS medium, 2~ of sucrose, 0.5 mg/l of benzylaminopurine BAP) and kept in the culture room in 12 hou:rs of light (3000-5000 lux) at ~4C. The ~ther half are propagated as a callus culture on LS medium with 1 mg/l of NAA, 0.2 mg/l of kinetin, 0.1 mg/l of ~AP and 100 mg~l of kanamycin sulphate. When the regenerated shoots are about l cm in size, they are cut off and placed on 1/~ IJS medium (1% of sucrose, 0.8~ of agar), without growth resulators, for rooting. The shoots are rooted on 1/2 MS medium with 100 mg/l of kanamycin Le A 28 417 - 26 -. . .. .. . _ . .. _ _ .~ ~ _ ,. . . . ...
_ . . .. _ . _ . . .

2~7~7 sulphate and later transferred into soil.

g) Transformation of leaf discs by Agrobacterium tumefaciens_ _ _ For transformation of leaf discs (Horsch et al.
1985), leaves about 2-3 cm long from sterile shoot cultures are stamped into discs of 1 cm diameter and incubated with a suspension of appropriate Agro-bacte~ia (about 109/ml) (compare c~ in Am medium, see below) for about 5 minutes. The infected pi~ces of leaf are kept on MS medium (see below) without hormones for 3-4 days at about 24C. During this period, Agrobacterium grows over the pieces of leaf.
The pieces of leaf are then washed in MS medium (O.5 mg/ml of BAP, O.1 mg/ml of NAA) and placed on the same medium (O.8~ of agar) with 500 ~.g/ml of cefotaxime and 100 ~.~/ml of kanamycin sulphate. The medium should be renewed after two weeks. Trans-formed kanamycin-resistant shoots are visible after a further 2-3 weeks.

Biochemical detection_method of transformation Neomycin phosphotransferase (NPT II) enzyme test:
;

NPT II activity in plant tissue is detected as follows by in situ phosphorylation of kanamycin as described by ReiB et al. (1984) and modified by Schreier et al. (1985). 50 mg of plant tissue are homogenised on ice in 50 ~1 of extraction buffer (10% of glycerol, 5~ of 2-mercaptoethanol, 0.1% of SDS, 0.025~ of ~romophenol blue, 62.5 mM Tris Le A 28 417 - 27 -2~73~7 pH 6.8), with addition of glass powder, and centrifuged for 10 minutas in an Eppendorf centrifuge at 4C. ~0 ~sl of the ~upernatant are applied to native polyacrylamide gel (145 x llO x 1.2 mm; separating gel~ 10~ of acrylami~e, 0.33% of bisacrylamide, 0.375 M Tris pH 8.8, collecting gel:
5% of acrylamide, 0.165~ of bisacrylamide, 0.125 M
Tris pH 6.8) and subjected to electrophoresis overnight at 4C and 60 V. As soon as the bromophenol blue marker runs out of the gel, the gel is washed twice with distilled water for 10 minutes and once for 30 minutes with reaction buffer (67 mM
~ris-maleate, pH 7.1, 42 mM MgCl2, 400 mM ammonium chloride). The gel is placed on a glass plate of the same size and covered with a layer of 40 ml of 1%
strength agarose in reaction ~uffer whieh contains the suhstrates kanamycin sulphate (20 ~g/ml) and 20-200 ~Ci of 3ZP ATP (~mersham). The sandwich yel is incubated for 30 minutes at room temperature and a sheet of phosphocellulose paper P81 (Whatman) is then laid over the agarose. Four layers of 3 MM
filter paper, ~Whatman) and a few paper handkerchiefs are stacked on top. The transfer of radioactive kanamycin phosphate phosphorylated in situ onto the P81 paper is stopped after 3-4 hours.
The P81 paper is incubated for 30 minutes in a solution of proteinase K and 1~ of sodium dodecyl sulphate (SDS) at 60C and then washed 3-4 times in 250 ml of 10 mM phosphate buffer pH 7.5 at 80C, dried and autoradiographed for 1-12 hours at -70C
(XAR5 film rom KodaX).

4. Transformation of Solanum tu~erosum tpotato) Le A 28 417 - 28 -2~73~
The transfonmation was carried out in exactly the manner described in EP-A-0,242,246, pages 14 to 15, the Agrobacteria containing Ti plasmids which carry the CCoAMT gene or the CCoAMT genes.

All the percentage data in the above examples relate to percentages by weight, unless stated otherwise.

The presence of the CCoAMT genes in the plant cells and plants (tobacco) obtained according to the above examples was confirmed by Southern blot analysis. The expression of the CCoAMT genes was detected by Northern blot analy-sis, and CCoAMT was detected with the aid of specific antibodies.

Some of the media employed in the transformation of plants and plant cells are descrihed below:

Am medium 3.5 ~ of X~HP04 1.5 g of KH2PO4 0.5 g of Na3 citrate 0.1 ~ of MgSO4 x 7H2O
1 g of (NH4)2SO4 2 g of glucose to 1 1 Medium for sterile shoot culture of_tobacco Macroelements 1/2 of the concentration of the MS salts Microelements 1/2 of the concentration of the MS salts Fe-EDTA ~urashige and Skoog (MS) Le A 28 417 - 29 -2 ~
Myo-inositol 100 mg/l Sucrose 10 mg~l Agar 8 gll Vitamins Ca panthotenate 1 mg~l Biotin 10 mg/l Nicotinic acid l mg/l Pyridoxine l mg/l Thiamine 1 mg/l pH 5.7 before autoclaving K3 medium For culture of Nicotiana tabacum petit Havana SRl, Nicotiana tabacum Wisconsin 38 and Nicotiana plumagini-folia protoplasts (Nagy and Maliga, 19763 Macroelements NH4NO3 250 mg/l KNO3 2500 mg/l caCl2-2H2O mg/l ~gSO4-7H2O 250 mg/l NaH2P04 lH2O150 mg/l (NH4)2SO4 134 mg/l CaHP04-lH2O mg/l Microelements H3BO3 3 mg/l MnS04 lHzO; 10 mg/l ZnSO4 4H2O 2 mg/l RI 0.75 mg/l ~a2MoO4-2H2G 0.25 mg/l CuS04 5E20 0.025 mg/l CoCl2 6H2O 0.025 mg/l Fe-EDTA Na2EDT~ 37.2 mg/l FeSO4-7HzO 27.8 mg/l Le A 28 417 - 30 -2~7~
Inositol 100 mg/l Sucrose 137 g/l (= 0.4 M
Xylose 250 mg/l Vitamins Nicotinic acid 1 mg/l Pyridoxine 1 mg/l Thiamine 10 mg/l Hormones NA~ 1~0 mg/l Kinetin 0.2 mg/l pH 5.6 Stexilise filter Linsmaier and Skooq_medium ~Linsmaier and Skoog 1955) For culture of regenerating protoplasts and for tissue culture of tobacco tumours and callus. Linsmaier and Skoog (LS) medium is Murashige and Skoog medium (Murashige and Skoog, 1962) with the following modifica-tions:
- thiamine is weighed in at a higher concentration of 0.4 mgtl instead of 0.1 mg/l;
- glycine, pyridoxine and nicotinic acid are absent.

Macroelements NH4NO3 .t650 mg/l RNO3 1900 mg/l CaClz 2H2O 440 mg/l MgSO4-7H2O mg/l 2S KH2PO4 170 mg/l Microelements H3BO3 6.2 mg/l MnSO4 1H2O22.3 mg/l znSO4 4H2O8.6 mg/l KI 0.83 mg/l Na2MOO4-2H2o0.25 mg/l Le A 28 417 - 31 -.

, _ _ _ . , . _ , . , _ . . _ . .

~2~&~3~ ~
CuSO4-5H2O 0.025 mg/l Cocl2 6H2o 0.025 mg/l Fe-EDTA Na2EDTA 37.2 mg~l FeSO4 7H23 27.8 mg~l S Inositol 100 mg~l Sucrose 30 g/l Agar 8 g/l Vitamins Thiamine 0.4 mg/l Hormones: NAA 1 my~l Kinetin 0.2 mg~l pH 5.7 before autoclaving The following literature can be cited for transformation of plants and plant cells:

Eraley R~To ~ Rogers S.G., Horsch R.B., Sanders P.R., Flick J.S., Adams S.P., Bittner M.L., Brand L.A., Fink C.L., Fry J.S., Fallupi G.R., Goldberg S.~., Hoffmann N.L., Woo S.C. (1983). Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80~4803-4807.

Fromm ME, Taylor LP, Walbot V (1986) Stable transforma-tion of maize after gene transfer by electroporation.
Nature 319: 791 793 Hain, R., S~abel, P., Czernilofs~y, A.P., SteinbiB, H.H., Herrera-Estrella, L., Schell, J. (1985~ Uptake, integra-tion, expression and genetic transmission of a selectablechimeric gene by plant protoplasts. Molec Gen Genet 199:
1~1-168 Hernalsteens JP, Thia-Tong L, Schell J, Van Montagu M

Le A 28 417 - 32 -2~73~ 7 (1984) An Agrobacterium-tranSform~ed Cell culture ~rom the monocot Asparagus officinalis. E~O J 3:3039-3041 Herrera-Estrella L., De Block M., Messens E., Hernal-steens JP., van Montagu M., Schell J. (1983) EMBO J. 2:
~87-995.

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A sLmple and general method for trans-ferring genes into plants. Science 277: 1229-1231 Krens FH, Molendijk L, Wullems GJ, Schilperoort RA (1982) in vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74 Koncz C, Schell J (1986) The promotor of TL-DNA gene 5 controls the tissue-specific expression of chLmaeric genes carried by a noval type of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396 Linsmaier DM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol plant 18: 100-127 Marton L, Wullems GJ, Molendijk L, Schilperoort PR (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium;tumefaci.ens. Nature 277: 1229-Nagy JI, ~aliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78: 453~455 Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn Le A 28 417 - 33 -;

2~73~ 7 B, Potrykus I (1984) Direct gene transfer to plants. EMBO
J 3: 2717-2722 Shillito ~D, Paszkowski J. Potrykus I (1983) Agarose plating and Bead type culture technique enable and stimulate development of protoplast-derived colonies in an number of plant species. Pl Cell Rep 2: 244-247 Van den Elzen PJM, Townsend J, Lee RY, Bedbrook JR (1985~
A chimaeric resistance gen as a selectable marker in plant cells. Plant Mol. Biol. 5, 299-302.

Van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR (1985) A chimaeric resistance gen as a selectable marker in plant cells. Plant Mol. Biol. 5, 299-302.

Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promotor ragment from the Ti Plasmid of Agrobacteri~m tumefaciens. EMBO J 12: 2723-2730 Van Haute E, Joos H, Maes M, Warxen G, Van Montagu M, Schell J (1983) Intergenic transfer and excharge recombination of restriction fragments clones in pBR322:
a novel strategy for the reversed genetics of Ti plasmids of /Agrobacterium tumefacines. EMBO J 2: 411-418.

Zambryski P, Joos H, Genetello C, van Montagu M, Schell J (1983) Ti-plasmid vector for the introduction of DNA
into plant cells without altering their normal regenera-tion capacity, EMBO J 12: 2143-2150.

Reiss, B., Sprengel, Will H./ and Schaller H (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell tracts r Le A 28 417 - 34 -20~73~ 7 GENE 1081: 211-217 Schreier P.H., Seftor E.A., Schell J. and Bohnert H.J.
(1985) The use of nucleAr-encoded sequences to direct the light-regulated synthesis and transport of a foreingn S protein into plant chloroplasts, EMBO J vol. 4, No. 1:

The following published patent applications may further-more be mentioned:

EP-A 116,718 EP-A-126,546 EP-A 159,418 EP-A-1~4,597 EP-A 120,515 EP-A-175,966 EP-A-120,516 WO 84/02913 EP-A-172,112 WO 84/02919 EP-A-140,556 WO 84/02920 EP~A-174,166 WO 83/01176 EP-A-122,791 The increased resistance of the transformed plants according to the invention may be illustrated with the aid of the following example:

Detection of the increased resistance of transformed plan~s _ Example A

To test for an increased resistance to plant diseases, the plants are inoculated with a pathogen and the degree of attack is used as parameter. Botrytis cinerea Pers.
is used as ~he test pathogen.

Le A 2~ 417 - 35 -2~$731 7 The tobacco plants are pregrown in tissue culture and subsequently potted in standard soil (Balster) in pots (d= 11 cm) in a greenhouse and grown in the greenhouse at 23C and 70-80% relative atmospheric humidity until the S start of the experLment. The plants are supplied with water and fertiliser as required. ~or inoculation, the leaves of the plants (3-4 weeks after transfer into the greenhouse) are sprayed with a spore suspension of the pathogen until dripping wet. The plants are then incuba-ted ~t 100% relative atmospheric humidity and 10-20C.
After 4-8 days, the state of health of the plants is determined in per cent with the aid of the leaf area attacked.

The transformed tobacco plants into which a CCoAMT gene according to the invention had been insertecl exhibit a significantly lower attack by B. cinerea than the of the non-transformed plants.

Explanations on diaqrams 1 to 3 (Fig. 1 to FiqO 3 !

Abbreviations used:

1 : Start of the encoding region 2 : End of the encoding region CaMV : Cauliflower mosaic virus CbR : Carbenicillin resistance gene E : EcoRI cleavage site H : HindIII cleavage site Km~ : Kanamycin resistance gene for plants P35S : CaMV35S promoter pA35S : Polyadenylation sequence of C~MV

Le A 28~417 - 36 -.. . . . .... ... .,. .. . _, 2 ~ 7 RV : EcoRV
S : SST1 cleavage site Arrow direction : Direction of the promoter and of the gene S LB ~ Left border sequence of the T-DNA
of A. tumefaciens RB : Right border sequence of the T-DNA
of A. tumefaciens Fig. 1: Fig. 1 represents a diagram of the plasmid pL2-4 which contains the protein-encoding sequence of the CCoAMT gene (compare also SEQ ID NO: 1) on the EcoRI fragment.

Fig. 2: Fig. 2 represents a diagram of the plasmid pRTlOl::CCoAMT which contains a chimaeric CCoi~NT gene.

Fig. 3: Fig. 3 represents a diagram of the plasmid pCVOOl::CCoiAMT which contains a chLmaeric CCoAMT gene.

Preferred hybridisation conditions As mentioned above, the preferred CCoi~T genes according to the invention are characterised in that they hybridise with the CCoAMT-cDNA 3equence contained in the plasmid pL2-4 or its components or with the cDNA sequence according to SEQ ID No: 1 or its components andencode CCoAMT.

Preferably moderate stringency conditions are u3ed. Moderate stringency conditions means preferably at 58 to 65C (particularly preferred at 63C) in 3 to 4 timesconcentrated SSC.

If this method is used to isolate CCoAMT genes from other sources, normally a population of cDNA's with related 3equence~ are obtained which can e. g. be expressed in E. coli. The enzyme activity can be determined (e. g. according to Pakusch et al, Arch. Biochem.
Biophys. 2-71 (1989), pp. 488 - 994) and the de~ired cDNA can be i~olated.
Le A 28 417 - 37 -20673~ 7 SEQ ID NO:l TYPE OF SEQUENCE: Nucleotide with corresponding protein SEQUENCE LENGTH: 1258 base pairs STRAND FORM: single strand S TOPOLOGY: linear TYPE OF MOLECULE: cDNA
ORIGINAL ORIGIN
ORGANISM: Parsley IMMEDIATE EXPERIMENTAL ORIGIN 0 NAME OF THE CELL LINE: Parsley cell culture (Petroselinum crispum) Features: from 1 to 370 BP 5' untranslated region from 371 to 1093 BP mature peptide from 1094 to 1258 BP 3' untranslated region PROPERTIES: cDNA for caffeoyl-CoA 3-O-methyltransferase ~rom parsley 1 csaSc.casg casat~cact taa.cagcta 2ccac.s2.s acc~.Sa2 , 51 ~ca- 5ca t'5ctgszc,~a ct.ca.c2gt cs2.Sats2. c..Sc-2 .
'~1 t52_5-2aS2 a..5.ct55a 2~t2ga2~s2 a2gscc2sc_ tccsrc2cc,2 1~' a-a2ctactg c.~cc~caa2 ctc5ss2ttt cc2t.c2s2_ a22c ,a22a ~01 tSasaatc2g cta22cs22c ttasg2se~2a 2sccsc'sa' .acta2at2.
2~1 zcaac ctgc atatgttcac t2t2ctaca cct2c.sc2- ctacaa2t,~
3~1 2c...t.sst tc2t'stsca cattct2tac at2cstt22g 2csccca..t ~51 gtcS.t.g,ca caaa-tcc2g 371 390 410 4~0 a~cgcttc~aatsc~ca2tcta2acattc2saasttg !~et~l2ce~r~snGiiGl~serLysnisscrGl~av2lGly:~isLysserLe~Lel~Gl~e`e-450 ~70 ~0 s2tsctc...a.cactatat2ctts2zac2a~ts'st2cccc2sasaacc252ssc2zt~
.as_.`.12TeuTirGlnlyrIleLe~GluThrSe_V2~yr?-cArs~ oC-l~ lz!'e.
513 5'0 j_~
22acacc.t2c2s2ac~tc2ccsca22Cca~cc2tss22tctcatsac2ccc.c2gc.~-z.
- j CGl~T e~ 5GluVai-~_.`.12Lig.:iisrroTr_~.s-~T e~ et~'~ r~er~!z~ ?

Le A 28 417 - 38 -2~731 7 570 ;o3 ~1 ~
c22ssgcasttcttc2acatccttttg2a5ctcatc22tgcca2a22cacc2t5c~ar-at .
Gl~GlyGlr~ e-e~:~.sr.!~etLe~euL~,~S-e~Ile~s.~l2Lys~.s~ e~cl~Ile ~ t.~ac_ct,~,t~a ,c,c,cc~tcccac.yccc~ ctc~ra-~ct--2-~-_23e~
5livai~ir~ -r~ly~y~rserLeu7-c~ al~ -!a;~ a C~-_c.`.c-.~.-j-~r-1;7 js 5~ 0 1;~
;`,r,C__~C-2~etc23c35aca2-----c~a~c-e2cti--c2-~2cc-__r_~._r2ee2a c!~- _ -- s? ~ 1 e~`~s r.~- 5G l ~ s ~ 2Gl~ ^, c ~ 1 2 -' ' 2~ S
750 7/~
_c-5~2st~-s~c-ca2a2ttcacttc2c~asa2ssccca~ctttscc~s~tcL~
AlaCiy'v2lr-` j:-.isL~sIle~isp?~.e~-gGl ~Gl~i?rc~la7e~-o~2~ s-~ s 810 a ~
a~rct -a2c,atc,saaast2tcatssaacatttc,2ttttstat t,'.sa.-,c' _~zac ! .~t_e ~C-l~_-.s?_l~ s L y r:- i sGl;~ r -~.e` s ~-~.c ;al -: C'~ 21 ` s -~ l - ;s-: s 37;) ac~
c,~a.aac.atc caactaCc2c2asac-..aatts2ttt2-,~aaaaatc,g_rc,,-c ._--,;s_,~,s_~ s~ s~s.'.rs_elIle.~.s~7~ 'v'al_isI'~Gl~ e 930 c,~0 ~ / ~
src-acS_c2ac-cC^ta.-gaat5rjttctstS~scbcasccasctsatsctcc-2~c~as2 r-li -l i~-'-S-~-s~ -;e~ ~ r_.`.s~.Gly~er~lal.~.la~:ls - -c.~.læ~.s^.~.la~-C,3 101~ 1~3 aac.c.s.a2_r.act2caS2cactt~stsa~t2Sic~t2ac2a2sc~ts~-ccs~r^r~;s~ 'al;-~.;,_.y_~rcis~-~e~zlllsC-l;l~e~s^_ys'la_~ `lc2'1_'s?
lu~ 10/3 lG,~
cccasc,zttgac_~c.,ct2tgcttcctc,ttsst2~gs2sttaccct-,'scc,.cc,.atc ~~cA_~lle5~ C!~s~ tLe'ù~ V21r-l~ s?Gl'j~21~r~'2' C'is.~.~c,.`.-c~1Q

ac,c _ _ _ ~ca.tatct2actc~22atttsagat2ttatttcac2ats.ttt2as22z~ss22t2c...
11 /0 1190 121 ~
tsc.t~c2ttc-atcttcctatgtttcttyttsaatttscaatctsca--at.c,a.~,a~g '2_~ 1250 12-a 2at2,2~tc2t2at.s2tstts2a22a2a2222aa2aa2a2222a Le A 28 41~ - 39 -

Claims (25)

1. Caffeoyl-CoA3-0-methyltransferase genes (CCoAMT
genes).

2. CCoAMT genes according to claim 1, which are characterised in that they hybridise with the CCoAMT-cDNA sequence contained in the plasmid pL2-4 or its components or with the cDNA
sequence according to SEQ ID NO:1 or its components, and encode CCoAMT.

3. CCoAMT genes according to Claim 1, obtainable from dicotyledonous plants.

4. CCoAMT genes according to Claim 1, obtainable from parsley.

5. CCoAMT genes according to claim 1 having a protein-encoding region which corresponds to the cDNA which is contained in the plasmid pL2-4, or shown in SEQ ID NO:1, and the DNA
sequences having essentially the same action.

6. CCoAMT genes according to Claim 1 which contain the TR promoter or the 35S promoter as promoter.

7. CCoAMT genes according to Claim 1 having a protein-encoding region which corresponds to the cDNA contained in the plasmid pL2-4 or shown in SEQ
ID NO:1, and having the TR promoter or the 35S
promoter.

8. Component of the CCoAMT genes according to Claim which has a regulatory action.

Le A 28 417-US - 40 -

9. Structural genes of the CCoAMT genes according to claim 1 and the DNA sequences which correspond to the cDNA contained on the plasmid pL2-4 or shown in SEQ ID NO:1.

10. Recombinant prokaryotic or eukaryotic DNA which contains one or more CCoAMT genes or their compon-ents as "foreign" DNA or as "additional" DNA.

11. Recombinant DNA according to Claim 10, which is contained in plant cells (including protoplastis) or plants (including plant parts and seeds).

12. Vectors which contain one or more CCoAMT genes or their components.

13. Vectors which contain the recombinant DNA according to claim 10.

14. Vector plasmid pL2-4.

15. Transformed microorganisms which contain one or more CCoAMT genes according to claim 1 or their components.

16. Transformed microorganisms which contain the recombinant DNA according to claim 10.

17. Transformed microorganisms which contain the sector according to claim 12.

18. Escherichia coli strain E. coli DS pL2-4 and its mutants.

Le A 28 417-US - 41 -

19. Use of the CCoAMT genes according to claim 1 and/or their components for the transformation of plant cells (including protoplasts) and plants (including plant parts and seeds).

20. Transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) which contain one or more CCoAMT genes and/or components thereof as "foreign" or "additional" DNA.

21. Transgenic plant cells including protoplasts) and plants (including plant parts and seeds) which contain the recombinant DNA according to claim 10 as "foreign" or "additional" DNA.

22. Process for the preparation of transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) having an increased resis-tance to pests, characterised in that (a) one or more CCoAMT genes according to claim 1 or their components are inserted into the genome of plant cells (including protoplasts), and if appropriate (b) complete transformed plants are regenerated from the transformed plant cells (including protoplasts) and if appropriate propagated, and if appropriate (c) the desired plant parts (including seeds) are obtained from the resulting transformed plants of the parent generation or further generations obtained therefrom.

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23. Use of the transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) according to claim 20 for the generation of propagation material and for the generation of new plants which contain the CCoAMT genes or their components and propagation material therof.

24. Propagation material, obtainable by propagation of the transgenic plant cells and plants according to claim 20.

25. Use of DNA sequences which correspond completely or partially to the cDNA which is contained on the plasmid pL2-4 or is shown in SEQ ID NO:1 for the isolation of CCoAMT genes from plants.

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